A number of scientific or patent publications describe methods for detecting cancer based on the identification of specific genetic alterations of circulating DNA or RNA.
U.S. Pat. No. 5,496,699 discloses a method for detecting mutations in nucleic acid sequences, in particular the sequence of the K-ras gene, in biological fluids such as blood, serum or plasma.
U.S. Pat. No. 5,068,175 discloses a method for detecting the presence of ras oncogene related malignancies in which said gene is quantified in serum or plasma samples.
WO01/42504 discloses the determination of extracellular nucleic acid, for example DNA of K-ras and APC genes, in serum or plasma samples, for the evaluation of the risk factor related with a number of neoplastic diseases.
WO02/18652 discloses a method of quali/quantitative detection of human telomerase RNA and telomerase reverse transcriptase RNA in plasma or serum for the diagnosis, monitoring, treatment or evaluation of a number of neoplastic diseases.
In principle, the methods for the molecular characterization of gene alterations have low sensitivity and require the analysis of a large panel of gene markers to obtain an acceptable information level.
Recently, a method has been reported for the quantitation of circulating naked DNA in plasma from lung cancer patients, based on a colorimetric assay able to discriminate between patients and healthy subjects and to early detect the relapse of the disease during follow-up (Sozzi, G. et al., Cancer Research 61, 4675-4678, Jun. 15, 2001). The colorimetric techniques (e.g. DNA Dipstick) for the quantitative evaluation of circulating DNA, however, are limited by a narrow linearity in the range 0.1-10 ng/ml, and by reduced sensitivity to lower values. In addition the test reading relies on subjective evaluation.
A more recent paper (Hsueh-Wei Chang et al., Journal of the National Cancer Institute, Vol. 94, No. 22, Nov. 20, 2002) suggested the analysis of DNA concentration in plasma samples and the analysis of SNPs for the detection of neoplastic disease, in particular ovarian cancer. DNA quantitation has been carried out by analysis of fluorescence intensity generated by the dye PicoGreen® linked to double-stranded DNA. The results show that, contrary to SNP analysis, the method used for measuring plasma DNA concentration is poorly sensitive and specific, and therefore is not suitable for the screening of population for neoplastic disease.